*2.11. Flow Cytometry Cell Analysis*

For GFP quantification, 2 × 10<sup>6</sup> parasites were harvested (1251 *g*, 10 min, 4 ◦C), washed once in PBS, fixed in 4% paraformaldehyde in PBS for 20 min at RT, washed twice in PBS, resuspended in 150 µL PBS, and immediately analysed by flow cytometry. The Cas9–GFP-expressing parental cell lines served as positive controls. The Cas9-expressing lines, which were negative for GFP, were included as negative controls to assess background fluorescence. Flow cytometric measurements were performed with the AccuriTM C6 flow cytometer (BD Biosciences, Heidelberg, Germany). A total of 30,000 events were recorded and analysed with FlowJoTM software V 10 (Becton, Dickinson and Company, Ashland, OR, USA).

#### *2.12. In Vitro Infection of Murine Bone Marrow-Derived Macrophages*

In vitro infections and parasite load quantification were performed as described [49–51].

#### *2.13. In Silico Procedures*

In silico cloning, DNA and protein sequence analysis were performed using the MacVector software version 17.x (Mac Vector, Cambridge, United Kingdom). Post-acquisition processing of images was performed using the ImageJ Fiji Software (Version 2.0.0, https://fiji.sc). Composite figures for publication were prepared using the Intaglio software (Purgatory Design, Durango, CO, USA). Numerical data and statistical differences were analysed using Prism (version 8, GraphPad Software, San Diego, CA, USA). Statistical comparisons between groups in the promastigote growth experiments were conducted using one-way analysis of variance (ANOVA)/Kruskal–Wallis test with Dunn's post

test. For comparison of intracellular parasite survival within macrophages, a ratio-paired, one-sided Student's *t*-test was applied to offset the variability between primary cell populations. Differences were considered significant at *p* < 0.05.

In silico design of primers to generate sgRNA templates and donor DNA was performed essentially as described [40]. Guide RNA sequences were designed using the Eukaryotic Pathogen CRISPR gRNA Design Tool (EuPaGDT, available at http://grna.ctegd.uga.edu) [52], using the default parameters (SpCas9: 20 nt gRNA length; PAM: NGG on 3' end; off-target PAM: NAG, NGA). In addition, two guide RNA sequences targeting *eGFP* (*eGFP*-52-5'sgRNA and *eGFP*-553-5'sgRNA) were retrieved from the Addgene repository (deposited as gRNA1 and gRNA2 by Guigo, Johnson; available at https://www.addgene.org/search/all/) as they had been experimentally validated for use in CRISPR experiments. Target-specific sgRNA primers were then designed manually and contained the T7 promoter (for T7 RNA polymerase-driven in vivo transcription of the sgRNA), the 20 nt sgRNA target sequence, and a sequence complementary to the sgRNA scaffold [17].

To generate gene replacement mutants, target-specific sgRNA primers were produced at http://www.leishgedit.net [17] (for whole GOI disruption) or designed manually (for partial GOI disruption). Donor DNA primer sequences contained target-specific 30 nt homology flanks corresponding to sequences immediately adjacent to the sgRNA target sequence for DSB-mediated repair by homologous recombination and recognition sequences for the pT template plasmids and were generated at http://www.leishgedit.net (for whole GOI disruption) or designed manually (for partial GOI disruption).

Since the sgRNA and donor DNA sequences identified using the EuPaGDT and LeishGEdit online tools used the *L. braziliensis* reference genomes (M2904 and M2903) available in TriTrypDB (https://tritrypdb.org/tritrypdb/), we verified the specificity of each sgRNA and homology flanks (donor DNA) by alignment against the *L. braziliensis* PER005cl2 genome [46] (focussing on chromosomes 20 and 29 which harbour the genes of interest) using the MacVector™ software ( Mac Vector, Cambridge, United Kingdom).

Oligonucleotides were ordered from Sigma-Aldrich (München, Germany). See Table S1 for a list of all primers.

## **3. Results**
